Custom RT-qPCR-array for glaucoma filtering surgery prognosis

<div><p>Excessive subconjunctival scarring is the main reason of failure of glaucoma filtration surgery. We analyzed conjunctival and systemic gene expression patterns after non penetrating deep sclerectomy (NPDS). To find expression patterns related to surgical failure and their correlation with the clinical outcomes. This study consisted of two consecutive stages. The first was a prospective analysis of wound-healing gene expression profile of six patients after NPDS. Conjunctival samples and peripheral blood samples were collected before and 15, 90,180, and 360 days after surgery. In the second stage, we conducted a retrospective analysis correlating the late conjunctival gene expression and the outcome of the NPDS for 11 patients. We developed a RT-qPCR Array for 88 key genes associated to wound healing. RT-qPCR Array analysis of conjunctiva samples showed statistically significant differences in 29/88 genes in the early stages after surgery, 20/88 genes between 90 and 180 days after surgery, and only 2/88 genes one year after surgery. In the blood samples, the most important changes occurred in 12/88 genes in the first 15 days after surgery. Correspondence analyses (COA) revealed significant differences between the expression of 20/88 genes in patients with surgical success and failure one year after surgery. Different expression patterns of mediators of the bleb wound healing were identified. Examination of such patterns might be used in surgery prognosis. RT-qPCR Array provides a powerful tool for investigation of differential gene expression wound healing after glaucoma surgery.</p></div

Microphotograph of preoperative conjunctival Impression Cytology (IC) with periodic Acid-Schiff-hematoxylin staining, obtained from a glaucoma patient.

<p>The nonsecretory epithelial cells are small and round and retain the intercellular junctions. The nuclei are large and N:C ratio is 1:2. The goblet cells are abundant, plump, and oval. Magnification 40x.</p

List of genes in blood samples showing expression differences at 15, 90, and 360 days after surgery.

<p>List of genes in blood samples showing expression differences at 15, 90, and 360 days after surgery.</p

Schematic representation of the fluctuations in Intraocular Pressure (IOP) as a result of surgical treatment, at different stages of the study.

<p>In the group of patients analyzed in the first study, there was only one case of failure, a year after the surgery (IOP of 21 mmHg). For the remaining patients in this group, the intervention was successful, with IOPs maintained below 21 mmHg, which is the established criterion for determining the success of the surgery. The surgical failure case shows a scarring area where there it had been a channel allowing the drainage of aqueous humor.</p

Workflow for PCR-Array analysis of glaucoma filtering surgery.

<p>Surgical failure was defined as an IOP ≥21 mmHg in the absence of ocular hypotensive treatment. In this study, we enrolled 11 patients (five patients with surgical failure and six patients from the first study).</p

Correspondence Analysis (COA).

<p>Distribution of genes that separate the group of patients with successful surgery (green dots) from the patients with surgical failure (red dots).</p

Correlation IOP-gene expression in blood and conjunctiva samples.

<p>Correlation IOP-gene expression in blood and conjunctiva samples.</p

Genes with significant expression changes in patients with surgical failure group in comparison with those of the success group.

<p>Genes with significant expression changes in patients with surgical failure group in comparison with those of the success group.</p

Bleb characteristics.

<p>Corkscrew vessels and microcysts.</p

Human Basal Tear Peptidome Characterization by CID, HCD, and ETD Followed by in Silico and in Vitro Analyses for Antimicrobial Peptide Identification

Endogenous peptides are valuable targets in the analysis of biological processes. The tear film contains proteins and peptides released by the tear duct mucosal cells, including antimicrobial peptides involved in the protection against exogenous pathogens; however, the peptide content of the tear liquid remains poorly characterized. We analyzed naturally occurring peptides isolated from human basal tears. Mass spectrometry analysis of endogenous peptides presents a number of drawbacks, including size heterogeneity and nonpredictable fragmentation patterns, among others. Therefore, CID, ETD, and HCD methods were used for the characterization of the tear peptide content. The contribution of DMSO as an additive of the chromatographic solvents was also evaluated. We identified 157, 131, and 122 peptides using CID-, ETD-, and HCD-based methods, respectively. Altogether, 234 different peptides were identified, leading to the generation of the biggest data set of endogenous tear peptides to date. The antimicrobial activity prediction analysis performed in silico revealed different putative antimicrobial peptides. Two of the extracellular glycoprotein lacritin peptides were de novo synthesized, and their antimicrobial activity was confirmed in vitro. Our findings demonstrate the benefits of using different fragmentation methods for the analysis of endogenous peptides and provide a useful approach for the discovery of peptides with antimicrobial activity